The concentration of soluble aluminium (Al) in the soil solution increases at low pH and the prevalence of toxic Al3+ cations represent the main factor limiting plant growth on acid soils. Citrate secretion from roots is an important Al-tolerance mechanism in many species including soybean. We isolated mitochondria from the roots of an Al-resistant soybean (Glycine max L.) cv. Jiyu 70 to investigate the relationship between citrate metabolism and Al-induced citrate secretion. Spectrophotometric assays revealed that the activities of mitochondrial malate dehydrogenase and citrate synthase increased and aconitase decreased with increasing of Al concentration (0–50 µM) and duration of Al treatment (30 µM Al, 0.5–9 h). Al-induced citrate secretion was inhibited by the citrate synthase inhibitor suramin, and enhanced by the aconitase inhibitor fluorocitric acid. Mersalyl acid, an inhibitor of a citrate carrier located in mitochondria membrane, also suppressed Al-induced citrate secretion. Transcript level of the mitochondrial citrate synthase gene increased in soybean roots exposed to Al, whereas expression of aconitase showed no significant difference. Expression of Gm-AlCT, a gene showing homology to Al-activated citrate transporters was also induced after 4 h in Al treatment. The Al-dependent changes in activity and expression of these enzymes are consistent with them supporting the sustained release of citrate from soybean roots.